Linking number is an invariant topological property of covalently closed, circular DNA (cccDNA) Because there are no interruptions in either polynucleotide chain, the two strands of cccDNA cannot be separated from each other without the breaking of a covalent bond.

If we wished to separate the two circular strands without permanently breaking any bonds in the sugar-phosphate backbones, we would have to pass one strand through the other strand repeatedly.

The number of times one strand would have to be passed through the other strand in order for the two strands to be entirely separated from each other is called the linking number.

Linking number

Topological states of covalently closed, circular DNA (ccc DNA)Conversion of the relaxed DNA (a) to the negatively supercoiled (b) form of DNA by the action of topoisomerase

Topoisomerases During replication, the unwinding of DNA may cause the formation of tangling structures, such as supercoils. The major role of topoisomerases is to prevent DNA tangling.Structure of the Topo I/DNA complex dsDNA Topoisomerase I

Types of topoisomerases There are two types of topoisomerases:

Type I produces transient single-strand breaks in DNA.

Type II produces transient double-strand breaks.

The type I enzyme removes supercoils from DNA one at a time.

The type II enzyme removes supercoils two at a time.

Although the type II topoisomerase is more efficient in removing supercoils, this enzyme requires the energy from ATP hydrolysis, but the type I topoisomerase does not.

The topo I of both prokaryotes and eukaryotes are the type I topoisomerase.

The eukaryotic topo II, bacterial gyrase, and bacterial topo IV belong to the type II topoisomerase.

In eukaryotes, the topo I and topo II can remove both positive and negative supercoils.

In bacteria, the topo I can remove only negative supercoils.

The bacterial topo II is also called the gyrase, which has two functions:

(a) to remove the positive supercoils during DNA replication,

(b) to introduce negative supercoils (one supercoil for 15-20 turns of the DNA helix) so that the DNA molecule can be packed into the cell. During replication, these negative supercoils are removed by topo I.

The bacterial topo IV belongs to the type II topoisomerase. This enzyme is involved in decatenation.

During replication, the introduced negative supercoils are removed by topo I.

Without topoisomerases, the DNA cannot replicate normally.

Therefore, the inhibitors of topoisomerases have been used as anti-cancer drugs to stop the proliferation of malignant cells.

However, these inhibitors may also stop the division of normal cells. Some cells (e.g., hair cells) which need to continuously divide will be most affected. This explains a noticeable side effect, the hair loss.

Type I topoisomerases can only catenate anddecatenate molecules if DNA strand has a nickor a gap.

This is because these enzymes cleave only one DNA strand at a time

(a)To remove supercoils. This involves a double-strand break (indicated by a short line), allowing the tangled segment to pass through. The break is then resealed. (b) To remove catenanes. The topo II makes a double-strand break in one DNA molecule (the blue one), allowing the other molecule to pass through. The break is then resealed.The function of topoisomerase type II

Type II topoisomerases can catenate and decatenate cccDNA by introducing a double-stranded break in one DNA and passing the other DNA molecule through the break

( c) Entangled long linear DNA molecules, generatedfor example, during the replication of eukaryotic chromosomes, can be disentangled by topoisomerase II.

(d) DNA knots can be unknotted by topoisomerase II.

DNA topoisomers can be separated by electrophoresis Covalently closed, circular DNA (cccDNA) molecules of the same length but of different linking number are called DNA topoisomers.

Even though topoisomers have the same molecular weight, they can be separated from each other by electrophoresis through a gel of agarose.

The more compact the DNA, the more easily it is able to migrate throughthe gel matrix.

Thus, a fully relaxed cccDNA migrates more slowly than a highly supercoiledtopoisomer of the same circular DNA.

Electrophoretic separation of DNA topoisomersRelaxed or nicked DNALinear DNASupercoiled cccDNAHighly supercoiled cccDNA

Mode of action of topoisomerases To perform their functions, topoisomerases must cleave a DNA strand (or two strands) and then rejoin the cleaved strand (or strands).

Topoisomerases are able to promote both DNA cleavage and rejoining without the assistance of other proteins or high-energy co-factors (for example, ATP) because they use a covalent-intermediate mechanism.

DNA cleavage occurs when a tyrosine residue in the active site of the topoisomerase attacks a phosphodiester bond in the backbone of the target DNA This attack generates a break in the DNA, whereby the topoisomerase is covalently joined to one of the broken ends via a phospho-tyrosine linkage. The other end of the DNA terminates with a free 3OH group.

The phospho-tyrosine linkage conserves the energy of the phosphodiester bond that was cleaved.

Therefore, the DNA can be resealed simply by reversing the original reaction.

The 3OH group from one broken DNA end attacks the phospho-tyrosine bond reforming the DNA phosphodiester bond.

This reaction rejoins the DNA strand and releases the topoisomerase, which can then go on to catalyze another reaction cycle.

Although as noted above, type II topoisomerases require ATP-hydrolysis for activity, the energy released by this hydrolysis is used to promote conformational changes in the topoisomerase-DNA complex rather than to cleave or rejoin DNA.

Phospho-tyrosinecovalet intermediateCleavage and rejoining of DNA using a covalent tyrosine-DNA intermediate

DNA synthesis at the replication fork

The overall replication process and synthesis of new DNA strands catalyzed by DNA polymerase

DNA helicases DNA polymerases are usually poor at separating the two base-pairedstrands of duplex DNA.

Therefore, at the replication fork, a second class of enzymes, called DNA helicases, catalyze the separation of the two strands of duplex DNA.

These enzymes bind and move directionally along ssDNA using the energy of nucleoside triphosphate (usually ATP) to displace any DNA strand that is annealed to the bound ssDNA.

Typically , DNA helicases that act at replication forks are hexameric proteinsthat assume the shape of a ring.

Like DNA polymerases, DNA helicases act processively, because they encircle the DNA, unwind multiple base pairs of DNA.

DNA helicases separate the two DNA strands of the double helixHelicase

Origin selection and activation by the initiator protein There are specialized mechanisms (initiator proteins) that assemble DNA helicases around the DNA in a cell.

Initiator proteins typically perform three functions during the initiationof replication:

First, these proteins bind a specific DNA sequence within the replicator.

Second, once bound to the DNA, they frequently unwind a region of DNA adjacent to their binding site.

Third, initiator proteins interact with additional factors required for replication initiation (e.g. helicases & SSBs), thus recruiting them to the replicator.

Function of the initiator proteins during the initiation of DNA replicationreplicator

Action of topoisaomerases at the replication fork

The composition of the DNA pol III holoenzyme

The trombone model for coordinating replication by two DNApolymerases at the E.coli replication fork

Finishing replication Completion of DNA replication requires a set of specific events.

These events are different for circular versus linear chromosomes.

For a circular chromosome, the conventional replication machinery can replicate the entire molecule, but the resulting daughters are topologically linked to one another.

Type II DNA topoisomerass are required to separate (or decatenate) daughter DNA molecules.

Topoisomerase II catalyzes the decatenation of replication products

Replication of the very ends of linear chromosomes cannot be completed by the replication machinery we have discussed so far.

The requirement for an RNA primer to initiate all new DNA synthesis creates a dilemma of the ends of linear chromosomes, called the end replication problem.

Prokaryotic cells use a protein priming protein instead of RNA primers,the protein provides the priming 3OH to initiate DNA synthesis.

Eukaryotic cells use the enzyme, telomerase to replicate their chromosome ends (telomeres).

Telomerase is a polymerase acts to extend the 3 end of its substrate.

The end replication problem

DNA ligase As each newly formed segment of the lagging strand approachesthe 5end of the adjacent Okazaki fragment (the one just completed),DNA polymerase I takes over.

The DNA polymerase I has two functions:

1- The 5 3 exonuclease activity of this enzyme removes the RNA primer of the adjacent fragment.

2- Fills the gap between the DNA fragments.

Finally DNA ligase joins the DNA fragments by the formation of Enz-AMPComplex which binds to the 5 phosphate of one fragment activating it, soIt is susceptible to 3 OH attack to form a phosphodiester linkage.

Thus, the two DNA fragments are ligated into one fragment.

Mode of action of DNA ligase